Intro to T Cells

Before I get into T cells, there is one important concept in biology and immunology that must be understood so that T cells will make sense. A lot of things will make sense after this. It is the concept of Cellular Adhesion Molecules. What are they? They are proteins on the surface of cells that allow them to bind and interact with other cells. Did you ever wonder why a liver cell stays in the liver and a heart cell knows to stay in the heart? This is Cell Adhesion Molecules (CAMs).

There are 3 families of CAMs we will want to understand with the cadherins, the selectins and the integrins. The first is Cadherins which bind cells together and to the ExtraCellular Matrix (ECM is a web of proteins which cells bind to). Cadherins hold cells in place so they don't move. They bind to each other or the ECM. This is important in cancer as loss of these Cadherins allow cells to Metastasize. It also provides contact inhibition so cells don't undergo mitosis unless it is necessary like a cell is destroyed by injury. The only cells in the body that move around are the blood and immune cells.

This brings us to the first key concept of T cells with Trafficking. When a tissue expresses the right CAMs for the T cells, they will be able to grab ahold by using their Selectins and Integrins like L-Selectin. This allows them to move out of the blood flow and into the tissue where the selectins are being expressed. When T cells are inactive, they tend to only express selectins and integrins that allow them to travel from Lymph node to circulation and back to other lymph nodes.

They tend to patrol the circulation and lymph systems. That is their job to look for any Antigen Presenting Cell (APC) that might have some pathogen to activate them. When inflammation occurs, there are cytokines that activate the selectins and integrins on the T cells. They get secreted into the tissue and blood that surround the area of inflammation. This causes a change in those endothelial cells and the T cells. These inflammatory cytokines like IL8 will cause the selectins and integrins on both the T cell and Endothelial cell to change. Now the T cell can bind to the Endothelial cell and get pulled through into the tissue where it is needed.

This concept of trafficking is an active process in immunology. It doesn't happen on its own. It requires inflammation and proper signaling to cause the change in expression of Selectins and Integrins on these cells. It also applies to other things like Vectors to deliver genetic therapies. A vector like Lipid Nanoparticles (LNP) can not traffic because they don't express the selectins or integrins to get out of the blood stream. Some things are small enough to diffuse across the blood vessels into the tissue in the capillaries where vessels are leaky. This happens with Complement proteins, Antibodies and Viruses that are so small they just pass through with the Osmotic pressure. Understanding how T cells traffick into tissues is a critical concept for both immunology and oncology.

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T cell Development

The Stem cells in the bone marrow produce all the cells of the circulation including the White Blood cells. There are about 10 immune cells that come from these Stem cells. We are only going to focus on the T cell here. The stem cells can give rise to 2 progeny in the Myeloid Progenitor and the Lymphoid progenitor. This happens by the stem cell during replication which creates 1 new stem cell and 1 new cell of the myeloid or lymphoid progenitor. This is called asynchronous replication. The T cells come from the Lymphoid progenitor cells.

The precursor to the adult T cell forms in the Bone Marrow. It will only have cellular adhesion molecules on it that will allow it to go to the Thymus. It will be released from the bone marrow where it will travel the circulation until it reaches the Thymus and its cell adhesion molecules will bind and allow it to enter the Thymus. When it enters the thymus, it will be known as a Double Negative T cell (DN). It will now be called a Thymocyte.

This new Thymocyte will undergo multiple stages of development in the thymus. The first is called the Double Negative stage (DN). It has 4 stages of this DN phase it goes through. It has to develop its T cell receptor (TCR) during this stage. Here the new Thymocyte will attempt to use the genes for the Alpha and Beta T cell receptor first. If it is successful, it moves on. If not, it will attempt to use the Delta and Gamma genes. It does this by first trying to do recombination with the Beta chain genes. If it gets a successful Beta chain arrangement, it will move onto the alpha chain. It can only do this with one of the 2 chromosome alleles. This is called allelic exclusion. This ensures each and every T cell has only 1 receptor and every receptor is the exact same on that T cell. About 95% of the Thymocytes will become Alpha/Beta T cells. The other 5% will become Delta/Gamma T cells and leave the Thymus immediately.

Once the T cell receptor is formed it will be displayed on the cell surface along with other parts of the T cell receptor Complex. This includes multiple CD3 proteins and the Zeta chain proteins. It will have multiple CD3 chains with Gamma, Epsilon, and Delta chains of the CD3. It will also include 2 zeta chains. All these CD3 and Zeta chains have intracellular domains that have these domains which become activated during the T cell receptor complex activation. These are called Immunoreceptor Tyrosine-based Activation Motifs or ITAMS. These are critical to the T cell activation once the TCR engages antigen.

At this point the T cell will have a successful receptor. It will also produce and display both co-receptors with CD4 and CD8. This is the only time in the T cell's life it will have both. At this point the T cell is called a double positive T cell as it is positive for both of these co-receptors. So now we went from Double Negative Thymocytes to Double Positive T cells.

The next stage of development will be the process of Positive Selection. Each and every T cell will test its receptor with the MHC I and MHC II proteins being expressed by the cells of the thymus. Those that react too strongly are killed off as they would be self reactive. Those that bind well to MHC I will stop producing CD4 and go on to just express CD8 co-receptor. They will be called CD8+ T cells now. Those that respond well to the MHC II will stop producing CD8 and only express CD4. They will be called CD4+ T cells. This is the process by which the T cells figure out their role in the immune response by being sorted into helper and cytotoxic T cells according to which MHC they work best with.

The final stage of selection is called Negative Selection. During this stage of training, the T cells will test their receptors against self protein antigens. There is a set of cells in the thymus which have special genes that allow them to express a huge range of self proteins like the T cells will encounter in the circulation. Each T cell will test its receptor toward these proteins. If they react to them strongly, they will be killed off. Some of them that react modestly to a self antigen will get signals to become T regulatory cells. These T regs will enter the circulation and prevent other T cells from reacting to the protein that matches their receptor. They become the police of the immune system.

Once the T cells pass Negative Selection, they will be released into the circulation where they can begin to travel from lymph node to lymph node patrolling for antigens that match their receptors. The process of T cell training inside the thymus is so extensive that only 5% or so of T cells survive the process. That means 95% of them don't. This process gives the T cells central tolerance for self antigens. In the circulation they will be naive T cells which have not encountered antigens yet. Once they encounter their antigen they will mature into effector T cells.

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AIRE Role

Inside the Thymus, there is the Medullary region. This is where negative selection occurs. The cells there are called Medullary Thymic Epithelial Cells (mTEC). They have this special transcription factor called AIRE. This stands for AutoImmune Transcription Element (AIRE). If you ever looked into genetics, each cell only has active genes it needs for its role. The heart cell only has genes active; it needs to be a heart cell. The liver cell only has genes active for its role as a liver cell. The AIRE transcription factor turns on all genes in these mTEC cells. This allows them to display a host of proteins that are self. This plays a critical role in the negative selection process.

All the thymocytes will test their T cell receptor against these self antigens. Those that have a high affinity toward that self antigen will get the signal to undergo cell death. Those that have low affinity will get passed into the circulation. The rare few that have an intermediate reaction can be converted into T regulatory cells (T regs). They can get the signals that induce that transition from naive T cell into regulatory T cell. Their job will be to protect the body should any reactive T cells escape the negative selection process. There is a set of genetic diseases that stem from defects in the AIRE gene. This leads to massive multi organ autoimmune disorders. In patients who have a genetic defect in the AIRE gene, they don't produce AIRE, or they produce a form of it that is not functional. This leads to no self proteins being presented by the mTEC cells. That means all T cells will be passed through negative selection.

You might be wondering how the T cells don't kill all those friendly bacteria and viruses that live in and on us. They don't get checked in the thymus as part of the negative selection process. This is where peripheral tolerance comes in. The T cell needs to have multiple signals to confirm its activation. The binding of its receptor to a matching antigen is only one of them. It requires cytokines and other receptors to be activated to turn the T cell on. Without these confirming 2nd and 3rd signals, the T cell will most likely fall into angery which means it does nothing. Eventually it will die from neglect. If the T cell binds to an antigen, but then gets other signals like TGF-Beta, it can be induced into a T regulatory cell. That new T reg cell will then stop responses by other T cells to that antigen. This is the process of peripheral tolerance. It gets exploited by cancer to induce T reg cells that promote tolerance to the tumor.

When a T cell is removed by death either in the thymus by negative selection or in the circulation, this is called clonal deletion. That specific genetic combination of genes that created that T cell receptor will be deleted along with the T cell.

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VDJ Recombination

The T cell will begin the arrangement of its Beta chain of the T cell receptor. This works in a competitive environment as both sets of genes from mom and dad compete to see which one is able to rearrange a successful Beta chain first. The genes for the Beta chain are located on chromosome 7. They are divided into 3 categories of the Variable genes, the Diversity genes and the Joining genes. There are about 60 different variable genes to choose from, usually represented as V1 to V60. There are 27 Diversity genes to choose from, represented D1 to D27. Then there are 6 different Joining genes to choose from represented as J1 to J6. The enzymes that regulate the recombination process will randomly choose 1 Variable, 1 Diversity and 1 Joining gene. There are many possible combinations of these genes. During this process all the genes that are not selected are removed and broken down. This leaves the T cells with just 1 choice for each of these VDJ genes left. Not every gene segment has to be removed in recombination. For example, if the RAG enzymes select V20 out of 60 possible gene choices, all the genes before that are left in the DNA from V1 to V19. The same happens with both the D and J genes. The genes that are left inside the DNA after the recombination will get spliced out when they are transcribed.

The first Beta chain to reach a successful arrangement and display it on the cell surface will win. The other genes for the opposite chromosome and the Delta/Gamma genes will all be inactivated. This is the process of allelic exclusion to ensure each T cell only makes 1 receptor. If the T cell fails to make a successful Beta chain for any reason with either allele, it will try to make a Gamma chain from both alleles of that gene. This acts like a rescue for the cell to try another receptor. About 95% of the T cells will have an Alpha/Beta receptor and 5% will end up with Delta/Gamma receptors.

The next step will be to rearrange the alpha genes which are on chromosome 14. The alpha chain has only 2 gene categories to draw from. They are the Variable and Joining genes. There are about 70 possible genes in the Variable category to choose from, represented V1 to V70. There are another 61 or so genes in the Joining Category to choose from, represented J1 to J61. The alpha genes have no Diversity category. The location for the Delta genes are actually inside the Alpha genes located between the Variable and Joining genes. A successful arrangement of the alpha chain removes the Delta genes so no possible arrangement of those genes can occur.

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Recombination Sequences

The genes for the Beta chain are located on chromosome 14. There are multiple copies of each Variable gene, Diversity gene and Joining gene. How does the process work to select just one each of these genes? Each and even copy of a gene from VDJ will have a small Recombination Signaling Sequence (RSS) next to it. It is often shown as small triangles pointing in the direction of recombination, but it is far more complex.

The RSS sequence is made of 3 parts with a heptamer which is 7 bases long, then there is the 12 or 23 base signaling sequence, and the last piece is the nonamer which is 9 bases long. The heptamer is always closest to the gene followed by the 12/23 segment then the nonamer. This creates the 12 and 23 rule. This means only a gene that has a 12 segment can be pasted to a gene with a 23 segment. The way it works is all the Variable genes have a 23 base RSS sequence, the Diversity genes all have a 12 base RSS sequence on both sides and the Joining genes all have a 23 base RSS sequence on them. This ensures that only a Variable can be pasted together with a joining and a diversity can only be pasted to the joining gene.

There are 2 enzymes called the Recombination Activating Enzymes 1 and 2. The RAG1 and RAG2 will land on these 12 and 23 RSS sequences. They will bend the DNA into a loop. This brings the 2 RAG enzymes next to each other. The DNA in between is cut and removed. Then a set of enzymes comes in and reconnects the ends of the DNA to bring the genes together. Let us assume the RAG1 enzyme lands on the RSS for Variable 40 gene. The RAG2 enzyme lands on the Diversity 10 gene. The RAG enzymes will pull those 2 genes together forming a big loop of DNA that was in between for the V41 through J9 genes. They get cut out and removed. The rest of the genes stay in place. The enzymes then repair the DNA so now the V40 gene is connected to the D10 gene. In the same way the RAG1 enzyme can land on the Diversity 10 gene and the RAG2 enzyme can land on the Joining 2 gene. They will loop together all the DNA from D10 to J1 and discard it. Now you have V40 attached to D10 attached to J2. This is how the RAG enzymes use the RSS sequences to do recombination of the Beta chain DNA. The Alpha chain only has the Variable and Joining genes so it is much easier to do recombination, but the alpha chain still uses these RSS sequences.

When this Beta chain is transcribed, it will turn the entire V1 through J1 genes into one long primary transcript. Only the V40, D10 and J2 will be the actual mRNA. The rest of any remaining genes gets spliced out. The last key enzyme in recombination is Terminal Deoxynucleotidyl Transferase (TdT). This key enzyme does the repair of the DNA after the RAG enzymes make their cut. There are several others that participate, but this is the key one. It plays a role in many DNA repair processes to paste DNA back together. It is an important enzyme to remember.

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T Cell Activation

Antigen Presenting Cells (APCs) will engulf pathogens or even get infected by them. They break them down into MHC class I and class II antigens. They display them on their cell surface as part of the MHC I and MHC II protein complexes. One of the concepts that still need to be addressed is that of which types of pathogens get presented on MHC I and which get presented on MHC II, and which type of immune response happens for each. The key differentiator is intracellular vs extracellular.

Those pathogens that invade cells will be broken down by the proteasome and presented on MHC I. Since the APCs are cells, they love to be infected and present these antigens. Intracellular pathogens include all viruses, some bacteria and some parasites. Many bacteria and parasites are extracellular with a few exceptions. The Mycobacterium like Leprosy and Tuberculosis will invade cells. Some parasites like Malaria invade cells. Any pathogen who invades a cell will need to be presented on MHC I. This often can lead to miss handling of some pathogens like Tuberculosis with a wrong response.

Any pathogens that stay outside cells like bacteria will get engulfed and presented on MHC II. The Antigen Presenting cells can present both MHC II to the CD4+ helper T cells and MHC I the CD8+ cytotoxic T lymphocytes (CTLs). This is often called cross presentation. One APC presenting to both T cells at once.

The T cell needs 3 signals to become fully activated. This acts like a safety to prevent T cells from being activated when they should not be. This helps prevent autoimmunity. The first is the antigen presentation to the T cell receptor. There could be millions of CD4 T cells in a lymph node, but only 1 of them will have the receptor that matches that specific antigen. Antigen binding to the T cell receptor will be the first signal. The second will come from the APC itself. When they have become activated from encountering pathogens, They will begin to display proteins on their surface called B7-1 and B7-2. These B7-1 and B7-2 proteins (CD80 and CD86) will make contact with the T cells Co-Stimulatory receptors called CD28. This gives the second signal. One other thing here. When the T cell is inactive, it displays CD28, but once it is active, it changes to CTLA4 which blocks further activation. The 3rd and final signal will come from cytokines released by the APC like IL-12 which will help activate the T cell. Those cytokines from the antigen presenting cell will also help direct the T cell to which type of response is needed. This helps the helper T cells differentiate.

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Helper T Cells

The Helper T cell is the commander of the immune system; it will take antigens presented from the APC and cytokine signals to figure out how to differentiate. In the presence of IL-12, it will go down the lineage of a Cell Mediated Response. In the presence of IL-4 and IL-5, it will go down the lineage of a Humoral response.

When the T cell becomes activated, it will begin to release IL-2. This is an autocrine and paracrine cytokine. It can activate the same T cell that releases it, and it can activate other T cells in the area. This IL-2 will be a second signal for cytotoxic T cells to activate if they see antigen presentation.

The helper T cell can take several pathways to differentiate. With IL-12 from the APC, they will turn into T helper Type 1 T cells. These Th1 cells will direct a cell mediated response toward pathogens that invade cells. They will release IL-2 to activate cytotoxic T cells and IFN-Gamma to activate Macrophages. These cells are the effectors of the cell mediated response.

Another pathway the helper T cell can take is that of the T helper Type 17 or Th17. This happens when antigen is presented with IL-4. That causes the T helper to turn into Th17 cell. They will release IL-4 and IL-5 to activate B cells. The B cells will mature into plasma cells and begin making antibodies which will attach to pathogens outside of cells. These antibodies are often linked with autoimmune disorders and inflammatory disorders.

Another pathway the helper T cell can take is that of the T helper Type 2 or Th2. This happens in the presence of IL-5. This creates the Th2 helper T cells which will activate B cells toward parasites and multicellular organisms. This works with IgE antibodies. It also plays a big role in allergies and allergic reactions. My study of T helper cells tells me there is a fine line between helper T cells releasing IL-4 and IL-5 that determines which response is triggered. More IL-4 released tends to activate B cells to produce IgM and IgG toward bacteria and extracellular pathogens. When the T helper produces a lot of IL-5, it pushes the B cells to make IgE which works for fighting parasites.

The last 2 pathways the T helper T cell can take are a bit unique. We already talked about how an activated T cell in the presence of TGF-Beta can turn into a T regulatory T cell. This is one possible fate a helper T cell can take. The other is that of a memory T cell. This happens automatically. As the helper T cells differentiate, some of them will become long lived memory T cells. They will hang out after the infection is cleared to prevent reinfection.

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T cell Response

The Cytotoxic T cell response is directed toward infected cells. This includes viruses for the most part with some rare bacteria and parasites. For the majority of responses, it will be against a virus which invades the cells. This could be a cold in the sinus, a flu in the lungs or even a virus in the GI tract.

There are sentry cells that line all these tissues which make contact with the outside world. They are the Antigen Presenting cells like macrophages and dendritic cells. These Sentry cells like macrophages and dendritic cells will engulf or even be infected with the pathogens. They will display the antigens on their surface using MHC I and MHC II. They will begin to release inflammatory cytokines like TNFa, IL-1, IL-6 and IL-8. Some will leave the tissue and head for the nearest lymph node. This is where all the T cells and B cells hang out.

The T and B cells travel through the circulation by the blood and enter the lymph nodes as they patrol the body looking for an antigen that matches their receptor. The APC will take the antigens to the lymph node and present them to T helper cells. The APC will present the MHC II antigens to the Helper T cells which will become Th1 helpers when activated. The APC will produce the co-stimulation by B71 and B72 along with cytokines like IL-12. For the Cytotoxic T Lymphocyte, the APC will present its MHC I antigens along with co-stimulation by B71 and B72. The CTL still needs cytokine support for activation. This will come from the helper T cell after it becomes active. It will produce IL-2 which will be the final activating signal for the CTL.

The Helper T cells act as the generals of the immune system. They direct the response by releasing cytokines to activate and direct other immune cells. The CTL is the killer T cell which wipes out infected cells. This makes it the soldier of the immune system. The CTL will undergo clonal expansion. That means the single CD8 T cell that is activated will clone itself over and over again by mitosis taking a few cells and making a whole army of millions to fight the infection. This takes time as the population of these CTL clones doubles about every 12 hours. This is what causes swollen and sore lymph nodes during infection. Once the clonal expansion is complete, both the helper and CTLs will leave the lymph node into circulation.

The macrophages in the tissue will release the same inflammatory cytokines like TNFa and IL-1 which will act on the nearby blood vessels. They will begin to express the cellular adhesion molecules like selectins and integrins. The IL-8 released by the macrophage will enter the bloodstream and activate the passing helper and cytotoxic T cells. They will then begin to express their selectins and integrins which will bind to the selectins and integrins of the blood vessel endothelial cells. This will allow the T cells to bind and home into the tissue. Notice the T cells don't go into the tissue unless called by inflammatory cytokines. This is a key obstacle in oncology for solid tumors in the tissue. Once the T cells are in the tissue, the CTLs can bind to any tissue cell that displays the antigen that matches its receptor. It will release perforins and granzymes which will kill that cell.

The helper T cells that enter the infected tissue will release cytokines like INF-gamma which will in turn activate the macrophages. They will then become active with their abilities to ingest and break down pathogens. The macrophage can also engulf and destroy infected cells.

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T cell Therapies

Now that we have covered many of the concepts of T cells, we can begin to look at how T cells have become living therapies against cancer. It all started with CAR-T therapies. I remember the early days of these programs as an investor. It was like many early experimental therapies. There were challenges, setbacks and eventually success. It all started with autologous CAR-T therapies, but what does that really mean?

When it comes to using T cells for cell therapies against cancer, there are 2 limitations to the T cells. The first is that cytotoxic T cells use MHC I. This limits the therapies to a single patient due to rejection. The second major flaw is that T cells only recognize amino acids of proteins. This limits the ability of these cells to only target proteins expressed in the context of MHCI, but what if we could give a T cell and B cell receptor?

The B cell receptor can target proteins, carbohydrates, lipids and even nucleic acids. It can be anything expressed on the cell surface. The most logical cancer to test this was B cell cancers as they display some very unique proteins on their surface like CD19 or BCMA. This makes them a really good target with a unique set of antigens not shared with other healthy tissues. The idea that these cancers are in the blood and lymph nodes makes them super easy as the T cells don't have to travel anywhere to find them.

So what they did was take an antigen binding domain of the B cell and put it onto the CD3 and zeta chains of the T cell receptor complex. This created a Chimeric Antigen Receptor (CAR). They could harvest T cells from the patient and edit them to insert this CAR for CD19 or BCMA. They would take ratios of CD4 and CD8 T cells from the patient's blood and edit them for this new CAR receptor. Since this was taken from the patient and returned to the same patient, these were Autologous therapies and rejection was not an issue. They had some learning curves with these therapies. High doses early caused cytokine storms which caused deaths in early trials. The cytokine storm was caused by too many T cells releasing all those inflammatory cytokines at one time. It led to a sepsis-like cytokine storm. Later they learned to treat these storms with IL-6 drugs, and lowering starting doses made them much safer.

The Autologous therapy was a huge success with 80% of patients having great responses for upward of a year. There were 2 major challenges to these autologous therapies. The first was the patients with blood cancers were often heavily pretreated and seldom had enough good T cells left to make a CAR-T therapy. The other major challenge was these cells took upwards of 3 to 4 weeks to manufacture. This is a long time for very sick patients.

The concept of Allogeneic CAR-T therapies was born. This would take T cells from a donor and use them in a recipient who needed them. This solved both of the 2 major challenges with autologous, but it added more challenges. Donor cells are easy to harvest and manufacture so they can be used in any person. Most patients have their immune systems wiped out with conditioning before these therapies which makes allogeneic not an issue, but it adds side effects and limitations. When the host immune system returns, these therapies get cleared at around 6 months. This brings up the first major challenge of Allogeneic therapies and that is the lack of durability due to clearance by the host immune system recovery at about 6 months or sooner.

The next fix would be to take these donor T cells and knock out the MHC I and MHC II genes so there could be no rejection by host T cells. Early data suggested this would work, but NK cells still killed them due to lack of MHC I. Next they discovered possible fixes for this issue by inserting HLA-E and HLA-G into the MHC I gene locus. Other approaches use the CD47 insertion to try to prevent NK cell killing of these T cells. The lesser MHC I proteins and CD47 still pacify the NK cells in early tests. The other option is to use a defense receptor on the T cells that would let them kill off any other cells of the patient's immune system that try to target them. They called this the Autoimmune Defense Receptor. There is no data so far for these programs testing new approaches to cloak the T cells from the host's immune system.

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The 3 T's of Solid Tumors

Here we are going to look at the major challenges for using T cell therapies in solid tumors. They are Targeting, Trafficking and Tumor Micro Environment (TME)

The first challenge to using CAR-T cells in solid tumors is the targets they select. When it comes to blood cancers, you have simple antigens like CD19 or BCMA. They are only expressed on B cells. You can wipe out every B cell and still have a healthy individual. They would require monthly Ig treatments to give them protection, but they could otherwise live full and healthy lives. If you try to pick an antigen in the solid tumors, many of them are shared with healthy tissues. They are just mutated forms of the healthy proteins. Many of the proteins in solid tumors are actually inside the cells and not even on the surface to target. This makes targeting a challenge to CAR-T therapies. Most CAR-T have stopped with few exceptions where they think they might have a clean target that's only expressed on a specific type of tumor. Most cell therapies in solid tumors have moved toward T cell therapies and away from CAR-T. These are therapies like Tumor Infiltrating Lymphocytes (TIL), matched HLA T cell therapies against public antigens, and personalized autologous T cell therapies with neoantigens.

The next major challenge for CAR-T in solid tumors is Trafficking. If you remember our coverage of T cells, they don't go into the tissue unless called. They stay in circulation and lymph nodes. This makes them great at taking out blood cancers like Multiple Myeloma, Leukemia and Lymphoma, but it is a challenge in solid tumors. Solid tumors tend to be in the tissue where T cells don't go unless called by inflammation. Most tumors learn to thwart the immune response so they can survive. This poses a huge challenge to get those T cells where they need to go. You could already have all the T cells you need to kill that tumor, but they just don't know it is there. Getting the T cells to activate and traffic to the tumor is a huge challenge. Some approaches to this is to take Tumor Infiltrating T cells and use them. They are T cells extracted from the tumor itself. That means they found it once so they should be able to find it again. Taking them and activating them toward the cancer antigens shows promise. Another approach now are therapies that get injected into the tumor which promotes inflammation which calls the T cells to the scene of the tumor. This gets them into the tumor responding to something else in the hopes they see the tumor once they are there and respond to it. This includes proinflammatory cytokines like IL-12, RIG or STING. The issue here is these tend to have some toxic systemic side effects.

The biggest challenge to CAR-T in solid tumors is the TME. The tumor lives and survives because it has to develop mechanisms to avoid immune detection. The tumors will express TGF-Beta which induces T regulatory cells. They will make the environment one of acceptance of the tumor. They will block activated T cells. This is bad for the cytotoxic T cell therapies no matter what kind they are. The tumor will also express proteins like PDL-1. This is a surface protein that blocks the activation of the cytotoxic T cells. The tumor learns to deactivate the T cell response toward itself. This is the major challenge.

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Tumor Infiltrating Lymphocytes

This type of therapy is used in metastatic tumors. This is a cancer where the tumor cells have spread from the primary tumor to other parts of the body. They do these therapies by removing one of the tumors. This brings up the first challenge as not all patients will have a tumor that can be removed for these therapies.

The tumor gets shipped to the lab where they extract the T cells that invaded the tumor. They break down the tumor to create cancer antigens. They mix the T cells into a mix of these tumor antigens and IL-2. If you recall, the cytotoxic T cells need IL-2 to activate them. This allows the T cells to activate and clonally expand. They can create a polyclonal army of T cells toward that specific tumor. This can be upward of 17,000 different T cell clones in TIL therapies. The challenge here is they activate these cells in the lab, but they do nothing for helping the tumor environment which was preventing the cells from responding in the first place.

The patient then undergoes lymphodepletion before they receive these new T cells. This clears out any T regulatory T cells before the therapy. The data for these therapies has been impressive with upward of 40% of patients responding. Some even have long term durable responses. This is mostly in Melanoma and Non Small Cell Lung Cancer (NSCLC).

The ability of these therapies to be mainstream is extremely limited. They are autologous. This means it is a long process of 3 to 4 weeks to extract the tumor, make the TIL, and put them into the patient as treatment. These treatments are one patient one therapy with a long manufacturing time in between.

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TCR Therapies

The Autologous approach takes T cells from the patient. It will then follow 1 of 2 different manufacturing approaches. The first will be to test patient T cells vs known public antigens like NY-ESO, MAGE-A4, or PRAME. The T cells that respond to these public antigens will get edited for some enhancements like to boost their affinity or improve activity of the T cell. Then they are clonally expanded into a large population for a treatment. Another approach is they will take patient T cells and just insert another TCR that encodes a T cell receptor toward the antigen they desire such as a neoantigen they get from sequencing the cancer DNA. They tend to take a few weeks to manufacture these types of therapies.

Often these therapies are placed back into the patient with lymphodepletion to clear out the regulatory T cells. They still have many challenges with TCR therapies. Either they have to be autologous which is long manufacturing or they have to suffer the rejection challenges of allogeneic therapies. Many of these therapies do donor to patient match in the allogeneic space. I think this space has too many challenges to make it a big enough opportunity to warrant investment.

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